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Prepare chemically competent E. coli cells

Goal

Make a large lab stock of chemically competent E. coli cells for routine plasmid cloning. We typically use NEB-Stable cells (ecAS4 of ecHP56) for cloning in our lab.

Materials

  • Transformation Buffer

    component amount alt reagent
    HEPES 236mg
    KCl 186mg
    CaCl2.2H2O 290mg 219mg CaCl2
    ddH2O to 100ml
    pH to 6.7 with KOH (need about 6-10ul of 10M KOH)
    MnCl2.H2O 840mg 1156mg MnCl2.4H2O
    • Filter through 0.2µ filter.
    • Make fresh and store at 4C.

  • SOB Medium

    component amount
    Yeast extract 5g
    Tryptone 20g
    NaCl 0.584g
    KCl 0.186g
    MgSO4 2.4g
    • Add 1L dH2O.
    • Adjust to pH 7 with conc. NaOH.
    • Autoclave 45 min liquid cycle.

Procedure

  1. Day 0: Streak NEB-Stable cells (ecAS4.1 or ecHP56) on LB plates and incubate at 37°C, 16-19 hours.
  2. Day 1: Start overnights of a single colony of the strain, LB, 37°C, 5ml, 200rpm, 12–17 hours.
  3. Day 2: Dilute overnights (1:5 to 1:20) into 5mL LB and shake in 23-25C waterbath, 200rpm, 5-6hrs (want these to be in approx log phase in the afternoon)
  4. Day 2: After 5-6hrs measure the OD600 and dilute into 1L SOB accordingly for 23C 200rpm Strong lab shaker in 2.8L baffled flask (need to sign up a few days beforehand).
    1. General benchmark: ecHP56 cells double once every ~1.32 hours at 23C, so if you want to harvest in 19-20hrs, then dilute into 1L SOB for a final OD600 of 0.00002 (~14.5doublings)
    2. Example: if you measured the culture's OD600 to be 1.0, then do: (vol)(OD600 1.0) = (1L)(0.00002) -> vol = 20uL of culture into 1L SOB at 23C 200rpm should reach OD 0.4-0.5 in 19-20hrs
    3. Note that the spectrometer's most reliable readings are between 0.1 and 1.0, so you will likely need to make dilutions prior to measuring
  5. Day 3: Check OD600 every half hour ~2hrs before you think they should be ready (e.g., if you diluted for 19-20hrs, check at 17hrs)
    1. Bring the cuvette/pipette to the incubator and take an aliquot for measurement quickly
  6. While waiting for OD600 to reach 0.4-0.5, prechill the following reagents, place in cold-room:
    1. 4 × 500ml bottles on ice
    2. transformation buffer on ice
    3. 2 x 50mL conicals on ice
    4. PCR strip tubes
    5. Set beckman centrifuge to 4C to prechill (include the yellow 50mL conical holders so they can prechill too)
    6. Prepare an ice+water bath large enough to fit the 1L flask of cells
  7. When culture hits OD600\~0.4—0.5 rapidly chill your cultures by placing them in the ice-water bath in cold-room for 10 min and swirling them occasionally.
  8. Aliquot the cells into the 4×500ml pre-chilled bottles (equilibrate the weights by eye, \~250mL each) and spin 3250g, 20min, 4°C. Pour off supernatant.
  9. Resuspend the pellet in 10ml ice-cold TB by pipetting, transfer to the 2 x 50mL conicals (20mL/conical). Incubate in ice-water bath for another 10 min. Make sure that the centrifuge is cold.
  10. Spin down cells 3250g, 20min, 4°C. Pour off supernatant.
  11. Resuspend cells in 1-5ml of ice-cold TB and combine them.
  12. Bring up the total volume to 16.6mL with TB, then add 1.2ml of molecular biology grade DMSO.
    1. Aliquot the DMSO at room-temp, then bring to cold-room just before adding or it will solidify
  13. Mix well gently by pipetting and leave on ice for 10 min.
  14. Aliquot 50uL/100μl into pre-chilled PCR strip tubes with individual caps and place immediately in −80°C box.

Checking transformation efficiency

  1. Mix 100ng of MG1655+pUC19 with 50µl of competent cells.
  2. Incubate on ice, 30 min.
  3. Heat shock 42°C, 45s.
  4. Incubate ice, 2 min.
  5. Recover in 500ul LB, 1 hour, 37°C, 200rpm in culture tubes.
  6. Dilute 10µl of recovered cells into 1ml of LB -> 1:100
  7. Dilute 10µl of above diluted cells again into 1ml of LB.
  8. Dilute 10µl of above diluted cells again into 1ml of LB.
  9. Plate 100µl of the three dilutions on pre-warmed carb100 plates and incubate overnight at 37°C.
  10. Count colonies and note the transformation efficiency below with the date as CFU / 1µg of pUC19 / 50µl of competent cells.

Results

  • Dilution used = 10uL transformants / 500 recovered vol * 100uL plated / 1000uL diluent = 0.002
Prep by Prep date cfu Results (NEB's equation = cfu / ug DNA / dilution ) Results (cfu / µg of pUC19 / 50 µl of competent cells) -80C Box
kchen July 2020 6 30,000 cfu / ug DNA 1.2 cfu / ug DNA / uL cells chemically competent e.coli
kchen Sept 2020 190 950,000 cfu / ug DNA 38 cfu / ug DNA / uL cells Chem. competent e.coli Box 2
ton/kchen Feb 2022 10,000 100,000 cfu / ug DNA 2000 cfu / ug DNA / uL cells Chem. competent e.coli Box 2